Cancer recurrence and treatment resistance represent the primary obstacles to current cancer therapies, with the underlying mechanisms largely unclear. In recent years, the hypoxic tumor microenvironment is regarded as one of the major causes mediating the treatment failures. Among the complicated tumor microenvironmental components, mesenchymal stem/stromal cells (MSCs) and their derivative stromal fibroblasts are currently recognized as master regulators in every step of cancer progression including primary tumor growth and secondary metastasis. Moreover, accumulating evidence suggests that MSCs and stromal fibroblasts also play critical roles in chemoresistance through their proteomic or metabolic changes elicited by drug treatments. However, little is known whether MSCs and stromal fibroblasts are involved in radiotherapy resistance. My preliminary results revealed a much less susceptibility of MSCs to ionizing radiation, compared to cancer cells. In mouse models of breast cancer and melanoma, in contrast to untreated MSCs, the irradiated MSCs exerted a robust potential in promoting primary tumor growth and lung metastasis of cancer cells. Through gene expression comparison, irradiated MSCs were shown to express high levels of osteopontin (OPN) and CCL-5. Deficiency of OPN largely abrogated the cancer promoting effects of irradiated MSCs. We therefore hypothesize that MSCs and their derivative stromal fibroblasts are key radiation-responsive stromal components to facilitate cancer progression, through overexpression of OPN, CCL-5 and other mediators. Those stromal features very likely lead to radioresistance or cancer recurrence after radiotherapy. To test this hypothesis, in the mentored phase (K99) of this award, I will first validate the function and molecular mechanisms of radiation-modulated OPN and CCL-5 expression on MSCs in the tumor promoting potential. Furthermore, I will extend the findings in MSCs to their derivative stromal fibroblasts and develop novel stromal markers (OPN, CCL-5 and possibly other factors) to predict the radiotherapy failure in breast cancer patients. All of these endeavors during my mentored phase will contribute to the proposed work in the R00 phase of this award-to develop effective therapeutics to treat clinical relapse/failure of radiotherapy in animal models. During this independent phase, I will attempt to establish radiotherapy resistance model and cancer relapse model in mice, followed by testing potential chemical inhibitors or neutralizing antibodies to prevent and treat these failures. This study combines my previous training in mesenchymal stem cell biology and immunology, and my current training in breast cancer metastasis, treatment resistance and radiation oncology, which brings me to a new field investigating the roles of MSCs and stromal fibroblasts in treatment resistance at my independent stage. The results will not only provide important information for our understanding of the basic stroma radiology, but also help to develop novel therapeutics to target stromal OPN, CCL-5 and possibly other mediators as adjutants to improve the efficacy of radiotherapy.